(NAS Colloquium) Links Between Recombination and Replication: Vital Roles of Recombination

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Colloquium on Links Between Recombination and Replication: Vital Roles of Recombination

Colloquium

DNA replication meets genetic exchange: Chromosomal damage and its repair by homologous recombination

Andrei Kuzminov*

Department of Microbiology, University of Illinois, Urbana-Champaign, B103, Chemical and Life Sciences Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801–3709

Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair.

Replication makes identical copies of chromosomes, whereas genetic exchange, working in the opposite direction, scrambles homologous chromosomes to create new combinations of independently arisen alleles. Enzymatic mechanisms are opposite, too (Fig. 1): whereas replication separates the two strands of DNA duplex to synthesize their complements, homologous recombination [in this case, by single-strand annealing (1)] removes the complements to reestablish the original pairing. And, yet, there is a hidden unity in the apparent divergence, according to the participants of the National Academy of Sciences Colloquium entitled “Links Between Recombination and Replication: Vital Roles of Recombination,” organized by Charles Radding (chair), Nicholas Cozzarelli, Michael Cox, Kenneth Marians, and James Haber and held at the Beckman Center of the Academy in Irvine, California, on November 10–12, 2000. The recent surge in works on interdependence of DNA replication and homologous recombination, conducted in experimental systems ranging from bacteriophages to mammalian cell lines, highlighted faltering replication forks as the connecting points between the two seemingly opposite domains of DNA metabolism. A replication fork falters when it encounters an unrepaired DNA lesion or when its progress is blocked by a DNA-bound protein. As it turns out, the main mechanism of repair of faltering replication forks in all domains of life operates via homologous recombination.

The ideas, that replication forks can falter whereas homologous recombination can repair faltering replication forks, are not new. In 1966, Hanawalt proposed a scheme of replication fork collapse at single-strand interruptions in template DNA (2). In 1972, Strauss independently suggested replication fork collapse at nicks and proposed breakage of stalled replication forks (3). In 1974, Skalka suggested that the cell uses homologous recom

Fig. 1. DNA replication vs. homologous recombination. Chromosomes are shown as double lines. Parental strands are filled; daughter strands are open. (A) A chromosome. (B) Chromosome replication has been initiated. (C) Chromosome replication is nearing completion. (D) Chromosome replication is complete. (E) Strand degradation in preparation for homologous recombination has started. (F) Strand degradation is nearing completion, whereas annealing of the complementary strands is going on.

bination to repair collapsed replication forks (4). In 1976, Higgins elaborated the mechanism of stalled replication fork resetting to its present form (5). Now the time has come to appreciate these ideas.

Current hypotheses represent replication fork faltering and repair as follows:

When a replication fork encounters a single-strand interruption in template DNA, it collapses, generating a double-strand end (Fig. 2 A → B → C). The idea of replication fork collapse is based on observations that single-strand interruptions in replicating chromosomes cause chromosome fragmentation (6, 7).
When a replication fork is stalled because of a block in template DNA, it regresses, forming a Holliday junction and extruding the newly synthesized DNA in a duplex (Fig. 2E → F). The replication fork structure can be restored by exonucleolytic degradation of the extruded duplex (ref. 8; Fig. 2F → A), or the

This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA.

Abbreviations: ssDNA, single-stranded DNA; SSB, ssDNA-binding protein; cccDNA, covalently closed circular DNA; RPA, replication protein A.

*	E-mail: kuzminov@life.uiuc.edu.

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